The proposed work concerns the control, and activation and catalytic mechanism of several metal-requiring enzymes. These enzymes are of increasing complexity and exhibit complex control mechanisms. Phosphoenolpyruvate carboxykinase activity is important in Diabetes and Sudden Infant Death Syndrome. The control of activity may be modulated by cation requirement or a modulator which affects protein structure. The nature of the control and activation of this enzyme will be studied by kinetics, NMR and protein modification studies. Enolase is inhibited by fluoride ion in the presence of phosphate and exhibits a peculiar cation requirement. The fluoride inhibition will be studied by high field NMR and by EPR. The cation interactions will be investigated by formation of mixed hybrids and a kinetic and NMR investigation of the activation and metal and ligand properties. Yeast pyruvate kinase is an allosteric enzyme which catalyzes an important control point in glycolysis. The relationship between ligand binding, structural changes and allosteric effects will be clarified. This enzyme will be studied by direct binding analysis, NMR studies, hybrid formation and kinetic analysis. The AS-PRT enzyme complex has demonstrated a complex cation requirement. The identification of the cation locations and function will be performed by NMR studies and mixed subunit hybrids. These studies will clarify several different types of enzymatic control manifested in various enzyme systems. The cations appear to play a crucial role in each of these systems and an elucidation of these mechanisms and requirements will describe on a molecular level the functions and malfunctions of crucial enzymes.